Abstract

Aluminium metal has important role in the field of medical sciences, but it has been found in various studies that Aluminium can cause numerous toxic effects. Studies have proven the strong affinity of the metalloelements for the sulfhydryl group (SH), present in reduced glutathione (GSH) and other biological molecules. In this context, the study about the possible interaction of Aluminium metal with glutathione in blood components was of interest, as an indication about the extent of Aluminium toxicity and the role of glutathione in the conjugation and detoxification of the metalloelement Aluminium metal. The concentration and time dependent effect of Aluminium metal on the level of GSH in plasma and Cytosolic Fraction in blood was investigated, following Ellman’s method. It was found that Aluminium metal causes a decrease in the GSH level, which is more pronounced with increasing concentration of Aluminium metal and with time incubation as well. The observed effect GSH concentration may be presumably due to production of oxidized glutathione (GSSG) or then due to Aluminium-Glutathione (GS-Al) conjugate formation

Keywords

Introduction

Glutathione mainly exist in glutathione reduced (GSH)
and glutathione oxidized (GSSG) in the body, among
which; the GSH is more in concentration than GSSG [1].
In mammalian cells, glutathione is the dominant nonprotein
thiol (Lu, 1999a). The glutathione level ranges
from 0.1mM to 10mM different human tissues [2]. Glutathione
is synthesized in most of the body cells and also
transported from the liver to various other organs of the
body [3, 4].

The most important function of GSH is the detoxification
of xenobiotics and drug by GSH conjugation reaction [5].
GSH binds to heavy metals, like zinc, mercury, cadmium
and copper and transport them to the liver, where they are
further detoxified and excreted [6]. The oxidation state of
Aluminium is 3+ [7] and GSH has electron donating property
[8]. Thus this study was performed to reveal the possible
intrection of Aluminium metalloelement with the
GSH in the plasma and cytosolic fraction of human blood.
In the aquous phase of blood GSH is present and Aluminium
has strong affinity for GSH. This affinity mainly exist
between Aluminium and sulfhydryl groups of GSH
[9]. This affinity can decrease the reduced form of GSH
in the blood,but with the depletion of GSH, glutathione
synthesizing system start making more GSH from cysteine
via the γ-glutamyl cycle but if GSH is usually not
effectively supplied, however, if GSH depletion continues
because of chronic metal exposure [9-11] then the pharmacological
benefits of the Aluminium metal being used
for the help of body defenses can be harmful in nature to
the body defense system . The following study makes a
design to see the effects of Aluminium Sulphate, in respect
of concentration and time, on glutathione level in
cytosolic fraction.

Methods

Preparation of Solutions

0.9% NaCl solution was prepared by dissolving 0.9gm of
NaCl in 100ml of water. 50 ml solution of Aluminium
(1mM) was prepared by dissolving 17.1 gm of Aluminium
in distilled water. Glutathione standard solution
(1mM)) was prepared by dissolving 30.74mg of GSH in
100ml of 0.1 N HCl. Di,thiobis, dinitrobenzoic acid
(DTNB) (1mM) was prepared by dissolving 39.9 mg of
DTNB in 100ml of buffer solution. Phosphate buffer(
0.2M) having pH 7.6 was prepared by mixing 42.2ml
of NaOH (0.2M) and 50ml mombasic potassium phosphate
solution (0.2M) and making the volume up to 200
ml with distilled water. pH was measured and adjusted by
using pH-meter (Accumet meter, Denver instrument
company USA).

Preparation and isolation of blood components

Isolation of Plasma

Three male volunteers with average age of 25.5 years who
were found of good health and free from any disease were
recruited. All three volunteers were student of Gomal
University (Pakistan). A written consent was taken from
each volunteer indicated they were non-smokers and not
using any drug during the study period. 12 ml fresh venous
blood was collected from each volunteer and treated
with 0.5mM Na-EDTA to prevent clotting then 1 ml of
blood was taken and mixed with of 1ml of each concentration
of Aluminium (100,200,300,400,500.600, 700,
800,900 and 1000 μM) solution and incubated for 10 minutes.
Then the solution containing equal volume of blood
and Aluminium in 1:1 ratio was then centrifuged at 1000
rpm for five minutes. The supernatant fluid was removed
with pasteur pipette, transferred to sample tubes kept on
ice till use and the remaining portion after taking
plasma was then processed for cytosolic fraction. Control
was also centrifuged for isolation of plasma and cytosolic
fraction containing equal volume of blood and 0.9% NaCl
solution and incubated as before.

Isolation of Cytosolic Fraction

The remaining cells were washed twice with isotonic saline
solution and blood cells were lysed at 40C with an
equal volume of distilled water for 1hour then after 1hr
cold mixture of chloroform-ethanol (3:5v/v) 0.8ml was
added to 2ml of lyed cells to precipitate the hemoglobin,
followed by addition of 0.3 ml of distilled water. Then the
mixture was centrifuged and pale yellow clear supernatant
was removed by pasteur pipette and transferred to sample
tubes and stored on ice till use. Control containing equal
volume of blood and NaCl solution was centrifuged as
before.

Determination of biological inorganic parameters

Plasma GSH (Extracellular)

Lysate GSH (Intracellular)

A standard Ellman’s method [12] was used for the estimation
of all glutathione as given below;
According to this method 2.3 ml buffer was added to
0.2ml of the sample (plasma or cytosolic fraction) followed
by addition of 0.5ml of DTNB. The mixture was
then analyzed by spectrophotometer. The reference cell
contained buffer. Blank consist of 2.5ml of buffer and
0.5ml of DTNB and measured against a reference cell
containing 3ml of buffer. All the measurements were carried
out at 412 nm after five minutes. Uv/visible spectrophotometer
of Model 1601(Shimadzu) was used for absorbance
readings.

Result

Effect of Aluminium on glutathione level of
extracellular plasma fraction of blood;

1ml of different concentrations of (100, 200, 300, 400,
500, 600, 700, 800, 900 and 1000μM) Aluminium was
added to blood in 10 separate tubes. Plasma in each tube
of blood was isolated after 10 minutes of incubation and
then transferred to separate tubes. Ellman’s method [12]
was used for the measurement of reduced GSH contents.
The plasma GSH level of blood decreased with the increasing
concentration of Aluminium as shown in the Results
are express as a Mean ± SE of 3 experiments of
plasma glutathione. There was found a significant change
in the content of plasma GSH from control was observed
at p<0.05 time intervals (0, 30.60,90,120,150,180 and 200
minutes for plasma GSH content was also measured when
two different concentration of Aluminium (100 and
1000μM) were added. The glutathione level of extracellular
plasma decreased at time intervals shown in the Figures
2-7. These results indicate that decrease in plasma
GSH level Aluminium concentration and time dependent.

Discussion

In the present study the 100-1000μM concentrations of
Aluminium was used. The main aim of this study was to
determine the effect of Aluminium metal on GSH level
present in both plasma (extracellular component) and cytosolic
fraction of the human venous blood sample. The
main focus was to explore the protective role of GSH in
the metals induced toxicity. GSH has variety of role as
antitoxin, free radical scavenger and participating in the
conjugation. Aluminium caused depletion of GSH
concentration in plasma and cytosolic fraction of human
venous blood. Depletion of GSH concentration in plasma
and cytosolic fraction could be due to the interaction of
reduced Glutathione and Aluminium either from oxidized
Glutathione (GSSG) or Al-SG complex. This study confirms the finding of other authors that concentration of
reduced GSH present in whole blood of human volunteer
is decreased by metal due to interaction of reduced GSH
and metal to form the oxidized Glutathione (GSSG) or
metals-SG complex [13]. The interaction between metal
and glutathione in vitro as model of in vivo reaction will
develop further scientific data and strengthen our knowledge
about the toxicological profile of Aluminium and the
protective role of Glutathione to prevent our body from
harmful effects [14]. Aluminium induced the depletion of
glutathione in blood components according to our findings.
There was found a positive correlation between the
exposer of GSH to the above given concentrations of
Aluminium and the depletion of glutathione with time (0-
200min).

The exact mechanism of the reaction between Aluminium
and depletion of reduced glutathione still need to be fully
investigated, but the formation of Al-GS complex may be
proposed. Considering the reducing power of GSH [8]
and the 3+ oxidation state of Aluminium [7], we can presume
a possible conjugation reaction between reduced
GSH and Aluminium metal. Although the exact mechanism
of this conjugation reaction cannot be distinguished
from this piece of research work, however this study can
be useful further to explore the role of GSH in the metabolism
and excretion of the important diagnostic agent
Aluminium. This research study also gives an idea about
the protective role of GSH against Aluminium toxicity.